The study of acute toxicity is a procedure in order to evaluate the adverse effects that might happen due to the administration of a single dose or multiple doses of a substance in a short period of time (24 hours). 14 Balb/c mice (7 males and 7 females) received KE-091/ATX orally in a single dose by means of cannulas adapted to syringes of 1ml graduated in 0.02 ml. The exact dose was calculated taking into account that the volume to be administered should not exceed 0.025 ml/g (25 ml/kg) of body weight. The dose that was given was 5,500 mg/kg. They were registered regarding time, duration and intensity. All mice were observed during the first hour and at 2,4, and 6 hours of post-administration. In the following 14 days, this was done 1 time a day. All animals that died during the observation period were registered. The principal signs of toxicity were observed in the one male (no females died) that died. Those signs were excitation, hemiplegia, eyes redness, dyspnea and clonic-tonic fits, which approximately started 15 minutes after the product was administered. The rest of the animals only developed excitation and transitory piloerection. Due to the immediate character and severity of the toxicity signs shown in the only mouse that died which are not characteristic of this kind of administration in substances with a similar solubility to the one present in KE-091/ATX, it was considered that this mouse presented a systemic absorption. It was not possible to identify ulcerations or any other damage in mucous epithelium during post-mortem examination.

Medium lethal dose was not obtained because of only one male animal died at 4 hours after a single oral dose larger than 5 g/kg. Substances with a LD50 larger than 5 g/kg orally administered to rodents are considered as non-toxic or moderately toxic substances. Thus, this pharmaceutical preparation of KE-091/ATX is practically non-toxic when orally administered.

Tests were performed to evaluate the possible irritant and toxic effects in skin and vaginal, rectal, and ocular mucosa in rabbits.

Vaginal Irritability: 18 Female virgin albino F rabbits from CENPALAB were used, with a body weight of 2 and 2.5 kg and were equally divided into three groups:
1) Administered with KE-091/ATX; 2) Placebo administered; 3) Control without treatment. There was no irritation at the level of vaginal mucosa since it is within the range of minimum irritation.

Rectal Irritability: 18 male albino F rabbits were used with a body weight of 2 and 2.5 kg and were equally divided into three groups: 1) KE-091/ATX suppository administered; 2) Placebo suppository administered; 3) Control without treatment. There was no irritation at the level of rectal mucosa since it is within the range of minimum irritation.

Dermic Irritability: 6 male albino F rabbits with a body weight of 2.5 and 3 kg were used. They were debilitated 24 hours before the test in the dorsal part of the trunk. The topical solution was directly administered from the flask, with any solvent in a volume of 0.5 ml at each site of assay. It was then covered with a porous gauze plaster of approximately 25 mm X 25 MM and mixed at the place with adhesive band during 4 hours. After this, the pastier was removed and each site was washed with distilled water; the site was marked for further observations; which were made at 1, 24, 48,72 hours after removing the pastier. The irritation degree was evaluated according to the classification system of Draize scale based on the apparition of edema and erythema observed at each administration site. There was no dermic irritation as the edema and erythema formation was non-existent.

Ophthalmic Irritation: 6 male albino F1 rabbits were used with a body weight of 2.5 to 3 kg and had received a thorough ocular examination with an ultraviolet light lamp. The topical solution was administered without any solvent in the conjunctival sack of the right eye in each animal in a quantity of 0.1 ml; the left eye was taken as a control. Both eyes of each animal were observed at 1,2,4,24,48,72,96 and 168 hours after the administration of the medicine. The evaluation was made by Garcia et al., method based on the Draize scale. Slight alterations were observed in the evaluated animals, but this irritation diminished during the course of the assay. The response degree obtained according to the classification system was 10.4. This value classifies the product as a slight irritant.

The purpose of the AMES test is to determine if a substance has mutagenic potential. The mutagenic effect is fundamentally due to the analogy of physical or chemical agents for nucleic acids and it may determine genetic mutations at sub-toxic concentrations. The genic mutation assay in bacteria, AMES Test, is the most frequently used for the evaluation of mutagenic properties in chemical products. Five strains of Salmonella typhimurium were used (TA 1535 HisG, TA 1537 His C, TA 1538 His D, TA 98 His D, TA 100 His G). At doses of 5,000 and 20,000 ug/pl, the product was toxic with the strain tested and after checking there was no mutagenic effect in the dose of 500 ug/pl, it was decided to test a dose range between 1,000 and 3,000 ug/pl in the final study.

The results of the tests show that KE-091/ATX does not show a mutagenic effect at any of the studied concentration for all strains included in AMES Test. Because of the phenotypic characteristics of Salmonella typhimurium stocks selected in the AMES Test, KE-091/ATX does not present a mutagenic effect of the kind of mutations due to a frame shift running nor to changes of base pairs. KE-091/ATX metabolites coming from the extrinsic enzymatic action (supernatant of homogenate of rat liver treated with Arocolor) do not present a mutagetic activity in any of the studied concentrations.

Though KE-091/ATX is a compound of anti-microbial action, it was possible to analyze its effects regarding retrogressive mutation in bacteria, at the interval of doses frequently used in Ames Assay (10 to 5,000 ug/plate). Toxic concentration values found in some strains do not correspond to the reported inhibitory concentrations for KE-091/ATX in several pathogenic bacteria.


Tests were performed to study the acute toxicity in order to evaluate adverse effects that might happen due to the administration of a single dose or multiple doses of substance in a short period of time (24 hours). 10 Balb/c mice (5 females and 5 males) with average weights between 20 and 24 g and age of 6 weeks were administered KE-091/ATX (1g/ml) topically in a single dose in the dorsal area of the animal. An area of approximately 2 cubic cm was previously shaved. 100 ul of neat KE-091/ATX was spread to each animal on the selected site. All mice were frequently observed the day of the administration (each 2 hours) and in the following 14 days 1 time a day.

No toxicity sign was observed during a period of 15 days in animals of both sex, after being topically administered with a larger dose of 5000 mg/kg. There was no sign of dermic irritation during the time of observation. Therefore, KE-091/ATX does not show any toxicity sign due to its topical administration in single doses to mice of both sexes.


Tests were performed to study the acute toxicity in order to evaluate the adverse effects that might happen due to the administration of a single dose or multiple dose of a substance in a short time period (24 hours) when it is intraperitonial (I.P.) administered. 42 (21 females and 21 males) Balb/c mice age 6 weeks and weighing between 20 and 24 g were split into 3 groups of 14 with half the mice being male and the other half female. The exact dose was calculated considering that the volume to be administered should not exceed 0.050 ml/g (50 ml/kg) of body weight. The first group received 1000 mg/kg, second 1500 mg/kg, and the third 2000 mg/kg. All animals were observed for the entire first hour after administration and afterwards every 2 hours and in the following 14 days once a day.

The main signs of toxicity found in the animals were difficulty in walking, piloerection and swollen abdomens in the ones that died. In previous studies, it was confirmed the absence of toxic effects in the solvent administered to mice using this technique. The Minimal Toxic Dose value of the solvent was 14,000 mg/kg of body weight. During necropsy of the dead animals, an oily deposit was observed in the abdominal cavity of a similar volume to the one administered (not absorbed). LD50 was calculated using the Probits method, with the mortality data obtained during the whole time of observation to be 1909.4 mg/kg for females with a confidence limit at 95% (17.29.6-2107.9 mg/kg) and 2307.6 mg/kg for males with a confidence limited of 95% (1011.4 – 5265.1 mg/kg). The LD50 obtained for KE-091/ATX administered to mice of both sex are characteristic of a moderately toxic substance, regarding the effects due to an acute administration. A slight sensitivity is observed in males, but it might be due to the number of animals used. The characteristic of late deaths after 48 hours elapsed, without any other dramatic sign of toxicity suggest that it may exist a controlled liberation of the active principle at intraperitoneal oily deposit or that the damages provoked by a single administration result in late consequences. The target organ was not known.

Micronuclei tests were performed in mouse bone marrow to evaluate if KE-091/ATX when administered intraperitoneally to Balb/c mice is able to produce chromosomic aberrations, if obtained a significant increase in the formation frequency of micronuclei in bone marrow erythrocytes. 20 male Balb/c mice 7 weeks old with an average weight between 16 and 24 g were treated with 2 administrations separated 24 hours a part and then were killed 6 hours after the second administration. The product was administered using the intraperitoneal technique using 1ml syringes and broken equally into 4 groups: a) no KE-091/ATX; b) 100 mg/kg positive control; c) KE-091/ATX 500mg/kg; d) KE-091/ATX 700 mg/kg. Each animal was killed by cervical dislocation and after this; both femurs were removed and mixed with 1ml of Bovine Fetal Serum, to extract the cells form the bone marrow. It was centrifuged at 1,000 rpm for 5 minutes and then the cell pellet was re-suspended. They were placed in plates previously washed with sulfocromic mixture and a solution of methanol and clorhidric acid at 5%. Two plates were made for each animal. Plates were fixed with methanol for 10 minutes. After 24 hours, they were dyed with Giemsa at 5% for 16 min. The species of Balb/c mice used in this study of micronuclei in mice bone marrow is sensitive to known mutagens and significantly yields response with an increase in micronuclei % and a decrease in proportion of polychromatic and normochromatic erythrocytes. It should be noted that the loss of nucleated cells is an indication of cytoxicity.

The intraperitoneal treatment with two levels of dosage of KE-091/ATX in solvent, corresponds to 2 to 3 times the therapeutic concentration, does not cause mutagenic effects when statistically evaluating the appearance of micronuclei and the erythrocyte composition of normo and polychromatic cells.

Tests were performed to determine if KE-091/ATX, when topically administered to Balb/c mice, is able to produce chromosomic aberrations if a significant increase is obtained in the formation frequency of micronuclei in erythrocytes of bone marrow. 30 female Balb/c mice weighing between 16 to 20 g that were 7 weeks old were divided into 3 groups of 10 and given 2 administrations, 24 hours apart, with dosages of a) control, b) 500 mg/kg, and c) 1000 mg/kg. The product was topically administered, in the dorsal region, previously debilitated in an approximate area of 4 cm.

An abrasion was made to half of the animals in each group in the site where the product was administered, following a recommended procedure to avoid dermic lesions. All the animals were killed 24 hours after the second administration by cervical dislocation.

After this, both femurs were removed and perfunded with 1ml of Bovine Fetal Serum, to extract the cells from the bone marrow. It was centrifuged at 1,000 rpm for 5 minutes and then the cell pellet was re-suspended. They were spread in sheets previously washed with sulfocromic mixture and a solution of methanol and clorhidric acid at 5%. Three replications were made (sheets) per animal. Sheets were fixed with methanol for 5 minutes, 24 hours after they were dyed with Giemsa at 5% for 16 minutes.

The species of Balb/c mice used in this study of micronuclei in mice bone marrow is sensitive to known mutagens and gives a significant response with an increase in the % of micronuclei and a decrease in the proportion of normo and polychromatic erythrocytes. The topical treatment with two levels of dosage of KE-091/ATX in solvent carrier, which corresponds to 2 and 5 times of the therapeutic concentration (and of 100 to 200 times the therapeutic dose), even in intact skin as in skin with abrasion, do no cause mutagenic effects when statistically evaluating the surging of micronuclei and the erythrocyte composition of normo and polychromatic cells. The carrier solvent, used as a control since it is also a carrier of pharmaceutical formulation for use in humans, does not present any mutagenic property for this method of administration and for the dose used.

The most recent study was an open label, randomized dose ranging clinical trial for efficacy, safety and tolerability of Trioxolane (KE091/ATX) in asymptomatic HIV-1 individuals with CD4 counts of 100-500 cells/ul of whole blood. In this study, 48 patients with proven HIV-1 status were recruited. The study consisted of three arms at buccal dose levels of 100mg (Group 1), 200 mg (Group 2), and 400 mg (Group 3) daily for a period of 4 months. The patients CD4+ T cell counts were divided into low and medium, 100-250 cell/ul and 251-500 cell/ul of whole blood respectively before random allocation to the 3-treatment dose levels. Efficacy was determined largely on the basis of changes in the levels of CD4+ T cells as well as those of viral loads. Toxicity was assessed by monitoring liver, renal, and bone marrow functions. Follow-up was done every two weeks. At each follow-up, all toxicity and efficacy assessments were done. Because of costs and the known slow pace of change, samples for the analysis of viral loads were taken every month, but those of two monthly intervals were analyzed. CD4+ T cell levels were determined every two weeks.

A total of 48 individuals were recruited into the formal study. Five of them could not complete the study for reasons not related to the medication. Two were due to relocation of their work place following posting by their employers; two were due to the frequent travels in and out of the country that could not offer sufficient time for regular follow-up and visits. One simply indicated that he was too busy to adhere to follow-up timetable. However, they still insisted that they wanted to continue with the medication. They were then given medication, but were withdrawn from the study.

A total of 43 out of 48 subjects had complete examinations (clinical) and full laboratory results for various measurements completed and assessed at baseline and at each of the eight follow-up visits up to 16 weeks. There were 17 patients in Group 1 of whom 14 completed the study, 16 in Group 2 of whom 15 completed and 15 in Group 3 of whom 14 completed the study.

CD4+ T cell levels showed better increase over the base line in those on daily doses of 100 mg. The levels tended to plateau during the third month of treatment at an average of 30% increase over the baseline. Some had increases of more than 60 percent over the baseline.

On the average, 50 percent of the patients on 100 mg had significant increase in CD4+ T cells while it was 47 percent in those on 200 mg daily dose and 14 percent in those on 400 mg daily dose. At this stage, it is difficult to tell whether the cells we are observing and registering at subsequent follow-ups are the ones that were observed at the beginning. Obviously, some cells will have undergone apoptosis while others will have achieved senescence and died. Others will have been recruited and matured.

Some cells have been observed to rapidly mature and strongly express CD4 markers on their surface upon starting treatment. These cells are neither naïve nor memory/activated. These can serve as prognostic cells, and which had been dubbed Koech’s Prognostic Cells (KPCs).

It was, therefore, planned to examine the sub-populations of CD4+ cells through the assessment of the level of maturity of these CD4+ T cells, as it was believed that they were at different stages of development. This was going to be achieved by using variously labeled monoclonal antibodies CD45 RO (for memory cells), CD45RA (for naïve cells) and CD4 PerCP (for possible identification of CD4_ cells which had been dubbed KPCs). These cells are the R3 or the CD4 DIM. It was now possible to confirm Koech's earlier observations that these cells were indeed real and could be identified and or isolated using CD45RO PE and CD45RA FITC. These cells are vital in the prognosis of response to treatment.

A reduction of 50 percent or more of the baseline in viral load was considered significant while an increase of 100 percent or more was considered significant in the other direction.

Trioxolane at 100 mg/day given in 4 divided doses had a good effect on the viral load reduction of 66.7% at 2 months. This degree in viral load reduction dropped by 33.3% by 16 weeks. Similarly, the observed significant reduction in log viral load dropped by over 50% at 16 weeks. At 200 mg, given in 4 divided doses, Trioxolane showed a reduction in viral load of 46% at 2 months. This degree of reduction dropped by 29% by 16 weeks. At 400 mg similarly administered, the viral load drop was only 30% at 2 months, but this drop was insignificant. At 16 weeks the number of patients with this minimal drop in viral loads rose to 64%. But only one person showed any significant drop in the log viral load drop at both 4 weeks and 16 weeks follow-ups.

In the case of weight gain, the lower dose showed a consistent increase between 1 month and 16 weeks follow-ups than any of the two high doses. The higher doses showed inconsistent changes in weight. No adverse effects have been encountered in any of the doses. The drug, KE-091/ATX when given in four equal doses at a daily dose of 100 mg, is able to significantly increase the levels of CD4+ T cells. From the foregoing, the data suggest that the lower dose appears to cause a better drop in viral loads than the higher dose. However, even this lower dose showed a better effect at 2 months than at 16 weeks. This suggests that a much lower dose than 100 mg per day might be what is required for a possible sustained viral load reduction. The drug is able to cause significant viral load reduction. The 100 mg daily dose was found to be better than 200 mg or 400 mg daily dose. Further follow-up led to the conclusion that a daily dose of 100 mg gave better results and hence, all follow-up as well as subsequent patients were put on 100 mg daily dose beginning August 2000. Follow-up continues. Some of the data have so far not been analyzed. Patients continue to receive the drug and funds are being sought for Phase III.

Because of great variation in the results arising from the determination of viral loads, necessitating repeats, future samples for viral load were collected for different time intervals, accumulated and run at the same time. This will reduce variations. The results obtained so far justify the need for facilitating wider use of this drug in the clinical management of HIV/AIDS.